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Germination of Bacillus thuringiensis var. israelensis spores in the gut of Aedes larvae (Diptera: Culicidae)
JOURNAL
OF INVERTEBRATE
Germination
PATHOLOGY
45, l-8 (1985)
of Bacillus thuringiensis var. israelensis Gut of Aedes Larvae (Diptera: Culicidae)’ CHRISTOPH
Spores
in the
ALY
Department of Entomology. University of Califovniu, Riverside, California, Received January 3. 1984: accepted July 30, 1984
92521
In laboratory experiments, germination and growth of Bacillus thztringiensis israelensis in the gut of Aedes uegypti and A. rexans larvae (Culicidae: Diptera) was observed. The number of spores and vegetative cells in the gut of fiving larvae and in cadavers was estimated by plating homogenized larvae on selective agar plates. The number of spores per gut increased in the first 40-140 min of exposure to a maximum, and decreased in the subsequent time, demonstrating spore germination in living larvae, moribunds, and in cadavers. Twenty-four hours after the death of the larvae, a minimal amount of spores, but an increased number of vegetative cells. was found in cadavers. In A. aegypti larvae, germination and growth of B. tlutringiensis israelensis in the larval gut was photographically documented. B 1985 Academic PESS, I~C. KEY WORDS: Bacillus thrrrinpiensis israelensis; Aedes aegypti: Aedes vexans: bacterial germination.
Bacillus thuringiensis var. israelensis was originally isolated from samples collected at a temporary mosquito breeding site in Israel (Goldberg and Margalit, 1977). Protein crystals, formed by the bacterium during sporulation, are a highly effective stomach poison for mosquito larvae (Tyrell et al., 1979; Lacey, 1984). Since toxicity is selective for Nematocera larvae (Schnetter et al., 1981), and resistance against the toxin remains low in mosquito populations (Vasquez-Garcia, 1983), B. thuringiensis israelensis is considered one of the most promising biocontrol agents in mosquito control (Anonymous, 1982). However, unlike classical biocontrol agents, which are characterized by their ability to recycle in the host and to suppress host populations over time, this pathogen is unable to establish high, lasting infections among natural mosquito populations (Ramoska et al., 1981; Vankova, 1982). Mosquito control requires frequent applications, which limits the importance of this agent (Service, 1983). In order to clarify the biological association between B. thuringiensis israe-
lensis and mosquitoes
as hosts, observations about the fate of this bacterium in the gut of Aedes larvae have been initiated and are reported here. MATERIALS
AND METHODS
Bottom soil and organic debris containing eggs of Aedes vexans were collected from natural mosquito breeding areas in the Upper Rhine Valley, Federal Republic of Germany, and were stored moist at room temperature in plastic bags. After flooding approximately 600 g of the soil and organic debris with 5 liters of tap water maintained at 30°C (Horsfall, 1956), 200-400 larvae hatched within 24 hr. The larvae were reared at ambient room temperature (20”-25°C). and were fed daily with Tabimin tish food (Tetra Co. ; composition Kardatzke and Liem, 1972). Random tests showed that more than 98% of the hatching larvae were A. vexans. A. aegypti larvae from a stock colony were reared at 22”-24°C on a yeast/dog food diet. Fourthinstar larvae were obtained S-6 days after hatching. All experiments were carried out using early 4th instars. B. thuringiensis israelensis. A freezeLarvae.
’ This work was partly supported by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 1
0022-2011185 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserves
2
CHRISTOPH
dried primary powder of a strain resistant to Rifampicin and Amphotericin B (Krieg et al., 1980) with a spore content of 4 x IO’ spores/mg was prepared. Test solutions were suspended with an ultrasonic blender (Braun Co.) and pasteurized (15 min, 7O”C), and a series of decimal dilutions was made in 0.25 mM phosphate buffer, pH 7.2. One hundred microliters of each dilution was mixed in a Petri dish with approximately 8 ml of liquid nutrient agar solution (Difco), containing 50 ppm Rifampicin and 4.0 ppm Amphotericin B. Colonies were counted after 18 hr incubation at 30°C. Using this method, the titer of a spore suspension could be estimated with an accuracy of to.3 x lox spores/ml. Cell titers were estimated with the same procedure, omitting ultrasonic suspension and pasteurization; in this case, colony counts represent the number of vegetative cells plus the number of spores of a suspension. All experiments were carried out in a continuously illuminated climate chamber. The experimental containers were covered with glass plates to prevent the disturbance of the water surface by the air current. In experiments I and 2, the larvae were kept without food 1 hr prior to the addition of the spore-toxin suspension. After the exposure to the test solution, the larvae were collected in a flame-sterilized metal-mesh basket and rinsed with tap water. In order to estimate the content of B. thuvingiensis israelensis spores and cells in the gut, the larvae were transferred into glass vials containing I ml ice-cold phosphate buffer, pH 7.2, per larva. In experiments 1 and 2, the larvae were homogenized with an ultrasonic blender. In experiment 3, the larvae were pulled to pieces with flame-sterilized forceps, and were homogenized by shaking the vial on a Vortex mixer (1 min, maximum speed). After taking an aliquot for estimating the cell titer in experiment 3, the suspensions were pasteurized, diluted, and plated as described above. Surface contamination of larvae. The number of spores adhering to the larval cu-
ALY
ticula after exposure to spore-toxin suspensions was estimated in the following experiment: groups of 10 larvae each were exposed to 100 ml ice-cold water containing lo4 spores/ml. At this temperature, larvae have no feeding activity. After 0.5, 1. 3, 6, and 24 hr, three groups of larvae were collected and washed as described above; each group was transferred to 1 ml ice-cold buffer. After vigorous shaking on a Vortex mixer, the number of spores present in the suspension was estimated as described. Experiment 1. To estimate the number of spores in the gut as related to time of exposure and water temperature, two l-liter glass containers were filled with distilled water and 100 A. vexans larvae each. One container was incubated at 19.5”C, the other at 27°C. After an acclimatization period of I hr, a spore-toxin suspension was added to final concentrations of 3 x IO3 spores/ml. After 20, 40, 60, 80, 100, 140 min, and after 3, 5, and 15 hr of exposure, the feeding activity of the larvae was checked by estimating the number of larvae still moving their labral brushes: 10 larvae were collected randomly to estimate the number of spores in the gut as described. The experiment was repeated with a sporetoxin concentration of lo4 spores/ml. Experiment 2. A series of beakers was filled with 100 ml water and 30 A. aegypti larvae each. By adding a spore-toxin suspension, a concentration of 1.9 X lo4 spores/ml was adjusted in all beakers. After 0.5. 1,2,4, 6,8, 12, 17, and 24 hr, mortality was recorded, and the larvae out of two beakers were collected to estimate the number of spores within the larvae. With exposure times of 0.5, 1, 2, and 24 hr, the experiment was carried out three times (six replicates). Experiment 3. To estimate the number of spores in the gut as related to the presence of food, 20 200-ml polystyrene beakers were each filled with distilled water and 20 A. vexans larvae. In 10 beakers approximately 20 mg wheat flour per beaker was added as a suspension. In all beakers, a
GERMINATION
OF
B. thuringiensis
spore-toxin concentration of IO4 spores/ml was adjusted; larvae were added after a lhr starvation period in clear water. After 20, 40, 60, 80, 100, 315, and 440 min and after 24 hr the larvae from one pair of beakers were collected. The larvae fed with wheat flour were placed individually on microscope slides; the amount of wheat flour in the gut, visible through the transparent cuticle, was examined under a dissecting microscope (method described by Dadd, 1968); each group of larvae was transferred to 1 ml buffer and homogenized. The titer of the homogenates was estimated as described. Optical observation of spore germination in gut. To suppress the growth of other bacteria in larvae killed by B. thuringiensis israelensis, sterile solutions of 50 ppm Rifampicin and 4.0 ppm Amphotericin B were used exclusively. To decontaminate the gut content, A. aegypti larvae were exposed for 1 hr to yeast cell suspensions in the antibiotic solution. Larvae were washed and transferred to suspensions of 50 mg B. thuringiensis israelensis primary powder1100 ml solution. The high concentration of primary powder was used to obtain a synchronous death of the larvae after ingestion of large and easy to observe amounts of spores. After 30 min of exposure, larvae were again washed, and their gut content was checked under the dissecting microscope as described. Control larvae were exposed to yeast cell suspensions and killed by decapitation. Larvae were transferred to aereated particle-free antibiotic solutions; 0, 6, 12, and 24 hr after death of the larvae, three larvae killed by B. thuringiensis israelensis toxin and three control larvae were collected and dissected; the gut content was checked under a phase-contrast microscope. RESULTS
Optical observation. After 30 min of exposure to 50 mg primary powder in 100 ml water, larvae were found hanging motionless at the water surface. Examination of
ismeiensis
IN Aedes
3
the gut content under the dissecting microscope showed that the larvae had nearly completely filled their gut with primary powder (4.6 & 1 gut segments filled out of a total gut volume of 6 segments). Under the phase-contrast microscope, dark-contrasted vegetative cells were already present in the gut besides bright-contrasted, ungerminated spores (Fig. 1A). Six, twelve, and twenty-four hr after the death of the larvae, masses of vegetative B. thuringiensis israelensis cells were observed in the esophagus (Fig. 1B) as well as in the gut (Fig. 1D). No bacterial growth was observed in control larvae up to 24 hr after decapitation (Fig IC). Surface contamination. After exposure to ice-cold spore suspensions, between 112 and 222 (mean = 179, standard deviation = 29) spores were found to adhere to the cuticula of one larva. This amount represents the number of spores not removed by the washing procedure, but resuspended by shaking larvae in buffer solution. No correlation with length of exposure time was found. Since this experimental situation was not identical with the situation in experiments 1-3, e.g., no cleansing activity by the larvae, data of experiments were not corrected with respect to the surface contamination data found here. Experiment 1. The feeding activity in the presence of B. thuringiensis israelensis primary powder suspensions was checked every 20 min in the first 5 hr of exposure. A concentration of 3 x lo3 spores/ml did not cause feeding to stop within this observation time. However, after 15 hr of exposure, all larvae had stopped feeding and the mortality rate totaled 58% (19.5’C) and 67% (27°C). A level of 1 x lo4 spores/ml caused a stoppage in feeding in the majority of the larvae after 160-180 min of exposure. The mortality rate totaled 15% (19.5C) and 27% (27°C) after 5 hr, and reached 100% at both temperatures after 15 hr of exposure. The spore content of the guts reached a maximum, in both spore concentrations and at both temperatures, 80- 100 min after
4
CHRISTOPH
ALY
FIG. 1. Bacillus tlzuringiensis israrlensis spores and vegetative cells in the gut of Ardes rwgypri larvae. Larvae were kept in solutions of antibiotics, and were exposed to 50 mg primary powder of an antibiotic-resistant 5. thwingiensis israelrnsis strain per 100 ml water. 100% mortality resulted after 30 min of treatment. Bars represent 10 urn. (A). After 30 min of exposure, vegetative cells (darkcontrasted) among ungerminated spores (bright-contrasted) can be observed in the gut. (B). Vegetative cells in the esophagus,. 12 hr after death of larva. CC). Gut of control larva, killed by decapitation and kept in antibiotic solution for 24 hr. Only yeast cells and no bacteria are present within the peritrophic membrane. (D). Gut of treated larva containing masses of vegetative 5. tlturingiensis israelensis ceils within the peritrophic membrane 24 hr after death of the larva.
GERMINATION
OF B. thrrringiensis
israelensis
IN Aedes
t IminI
t (min)
FIG. 2. Average spore content of 10 Aedes vexans fourth instars exposed to 3 x IO3 spores/ml (A) or IO4 spores/ml (O), at 19.5” (A) or 27°C (B). Five hours after treatment, larvae exposed to the lower spore concentration were still feeding. Spore content was estimated by plating homogenized larvae on selective agar plates.
the start of the experiment (Fig. 2). No difference between the numbers of spores present in the gut at two temperatures was observed when the same spore concentrations were offered. Larvae treated with 3 x lo3 (1 x 104) spores/ml showed a maximal spore content of 2.1-2.4 x lo3 (2.S 2.6 x 103) spores/larva after 80-100 min; the spore content declined to 1 .O- 1.2 x lo3 (2.0 x 103) spores/larva after 5 hr, and to 1.1-1.5 X 102(1.9 x lo*) spores/larvaafter 15 hr of exposure. In the surviving larvae treated with 3 x IO3 spores/ml for 15 hr, 7.0 X 102- 1.0 X lo3 spores/larva were found. Experiment 2. After 30 and 60 min of exposure, 1.3-3.1 and 3.6-7.3 x 10) spores per larva were recorded (Fig. 3). No change in behavior or feeding activity could be observed. Two hours after treatment, almost no feeding activity was observed; some larvae showed misorientated and ineffective swimming movements, but all larvae were still alive. Titers of 3.6-9.0 x lo3 spores/larva were estimated. After 4 hr, mortality totaled 95 ? 5%, and the number of spores per larva had dropped to 2.2-3.2 x 102. Up to 24 hr after treatment, similar numbers of spores were found in the cadavers.
Experiment 3. In this experiment not only the spore content but also the number of vegetative B. thuringiensis israelensis cells in the larvae up to 24 hr after treatment was estimated; additionally, feeding rates were recorded by measuring the amount of wheat flour ingested by the
IO4
0 0 III 12
I 4
I 6 Hrs.
I 8
I IO after
I 12
I 17
I 24
treatment
FIG. 3. Average spore content of 30 Aedes aegypti fourth instars exposed to 1.9 x lo4 spores/ml (T = 24-26”(I). Four hours after treatment, 95% mortality was observed. Living larvae were removed. Bars indicate standard deviation of six replicates.
6
CHRISTOPH
larvae. After 20, 40, or 60 min of exposure, larvae had 2.8 (standard deviation = 0.6), 5.1 (SD = 0.9), or 5.6 (SD = 0.7) gut segments filled with wheat, respectively. After 40 min of exposure, 6 out of 20 larvae had guts completely filled with wheat flour ( = 6 gut segments wheat filled). After 80 min, all larvae showed completely wheat-filled guts. In the absensce of food, the maximal spore content of I. 1 X lo4 spores/larva was observed 140 min after the start of the experiment (Fig. 4). In the presence of wheat flour, the maximum was observed 40 min after the start with 5.8 x IO3 spores/gut. Twenty-four hours after the start, 100% mortality was observed in both containers; 8.3-8.8 x lo? spores were counted within the larvae. However, 3.7-4.2 x lo4 vegetative cells were found in the cadavers (Table 1). This indicates not only spore germination, but also growth of vegetative cells in the dead host. DISCUSSION
Spores of B. thcwingiensis israelensis, ingested by Aedes larvae, germinate in the gut. After the death of the larvae, vegetative cells multiply within the peritrophic membrane. Approximately 1 day after
3
I 12
6
Time
(h)
FIG. 4. Average spore content of 20 Aedes t~exans fourth instars exposed to 10“ spores/ml, either with (0) or without (0) wheat flour (T = 22°C). Twenty-four hours after treatment, 8.3 x lo* (0) and 8.8 x lo* (0) spores/larva were estimated.
ALY
death, lysis of the larval tissues leads to mixing of gut contents with fluids in the hemocoel, resulting in the presence and growth of B. thuringiensis israelerzsis cells in all parts of the body. Time of spore germinution. Results indicate that germination of ingested spores occurs in living larvae, in moribunds, and in cadavers. In experiment 1, no stoppage in feeding was observed up to 5 hr after treatment with 3 x IO3 spores/ml. Nevertheless, the number of spores present in the gut declined after 80- 100 min of exposure. Defecation of spores is unlikely. since in A. vexans larvae the contents of the gut are not mixed by peristalsis (Aly, 1983). but are only pressed caudad and defecated when more food is ingested. Since the dry weight of the contents of one gut ranges between 100 and 200 pg, and only 0.75 kg (3 x lo4 spores/ml) and 2.25 )-Lg(lo4 spores/ml) primary powder/larva was offered in experiment I, replacement of the gut content, followed by defecation of the first ingested spores, is only possible if larvae feed continuously on their fecal pellets (Nilsson, 1983). In this case, the spore content of the gut should increase and remain on a constant level. Declining numbers of spores can only be explained with spore germination in the living larva. Decline in the number of spores in the gut also continued after stoppage in feeding and death, demonstrating a continuation of spore germination in moribunds and cadavers. Filter activity of larvae. The average filtration rate of A. vexans fourth-instar larvae is 3.3 t 1.1 ml hr-t larva-‘, when larvae are kept under the conditions of experiments 1 and 3 without food (T = 22°C 1-hr prestarvation; Aly, 1983). With a population density of 1 larva/l0 ml water, filtering during 160 min or more (exp. l), the number of spores suspended in the water would have declined during the exposure time due to filtration and ingestion. In these time frames, there was also a decline in the number of spores in the gut. This was due to both the decreasing number of spores
GERMINATION
Bacillus
thuringiensis
Time of exposure (hr) 1 2
5 24
OF B. fhuringiensis
isruelensis
TABLE 1 TOTAL CELLS AND SPORESIN THE GUT OF Aedes vexans
israelensis
Total cells/gut Without food
x x x x
3.7 x 104
103 lo3 103 lo4
LARVAE~
Spores/gut
With foodb 7.8 4.2 4.5 4.2
7
IN Aedes
With foodb 4.9 2.8 7.6 8.3
x x x x
Without food
103 103 lo? 10’
x x 6.1 x 8.8 x 6.6 8.9
103 lo3 lo3 lo2
n Twenty 4th-instar larvae in 200 ml of a suspension with lo4 spores/ml. b Twenty milligrams wheat flour/200 ml water.
suspended in the water and available for ingestion and to the continuing germination of the already ingested spores. Competition with other bacteria. Without antibiotics, the comparatively large cells of B. thuringiensis israelensis were few among enormous masses of other bacteria, also developing in the cadaver. Plating homogenized larvae without pasteurization on nonselective nutrient broth agar plates revealed up to lo7 bacteria per A. aegypti cadaver 1 day after death. Although unselective plating does not give a precise estimate of bacterial populations (Overbeck, 1968), the cadaver of a mosquito larva killed by B. thuringiensis israelensis toxin cannot be considered to be a relatively exclusive growth medium for this bacterium (Stahly et al., 1978). Nevertheless, B. thuringiensis israelensis was found to be able to multiply in Aedes larvae in competition with other bacteria. Sporulation and crystal production in the host. In the present paper, only germination and vegetative growth in the host have been described. From other experiments, resporulation and crystal production also seems possible: Larget (1981) and Ignoffo et al. (1981) observed a higher toxicity of B. thuringiensis israelensis suspensions when previously killed larvae had not been removed from the experimental containers. In A. aegypti (McIver and Siemicki, 1977) and A. vexans (Aly, unpubl.), larvae have been observed feeding on larval cadavers; this behavior may act as an important link
in the epizootiology of B. thuringiensis israelensis in natural mosquito populations. ACKNOWLEDGMENTS The author acknowledges the support of the present study by W. Schnetter, the technical assistance of B. Zimmermann, and the critical review of the manuscript of E. W. Davidson and C. Lucarotti.
REFERENCES ALY, C. 1983. “BeitrBge zur Ernahrungsbiologie der Larve von Aedes vexans.” Ph.D. Thesis, University of Heidelberg, Federal Republic of Germany. ANONYMOUS. 1982. Biological control of vectors of disease. Technical Report Series, Vol. 679, World Health Organization. Geneva. DADD, R. H. 1968. A method for comparing feeding rates in mosquito larvae. Mosquito News. 28, 226-230.
GOLDBERG, L.. AND MARGALIT, J. 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergeniii. Uranotaenia Culex uni~itattas, Aedes aegypti Mosquito News, 37, 355-358.
anguiculata,
and Calex pipiens.
HORSFALL, W. R. 1956. Eggs of floodwater mosquitoes III. (Diptera: Culicidae): Conditioning and hatching of Aedes vexans. Ann. Entomol. Sot. Amer..
49, 66-71.
IGNOFFO,C. M., GARCIA, C.. KROHA, M. J., FUKUDA, T.. AND COUCH, T. L. 1981. Laboratory tests to evaluate the potential efficacy of Bacillus thuringiensis var. israelensis for use against mosquitoes. Mosquito
News,
41, 85-93.
KARDATZKE, J.. AND LIEM, K. 1972. Growth of Aedes stimulans and Aedes vexans (Diptera: Culicidae) in saline solutions. Ann. Entornol. Sot. Amer. 65, 1425-1426. KRIEG. A., ENGLER, S., AND RIEGER, M. 1980. Produktion von Prgparaten auf der Basis von Bacillas tharingiensis var. israelensis mit UV inaktivierten Sporen zur biologischen Bekgmpfung von Stech-
CHRISTOPH
8
miickenlarven. Anz. Schiidlingskd. Pflun;. Umweltschutz, 53, 129-133. LACEY, L. 1984. Bacillus fhwingiensis serotype H-14 for the microbial control of mosquitoes. &r/l. Amer. Mosq. Contr. Assoc., in press. LARGET. I. 1981. &de de la rCmanence de Bucillus thuringiensis var. isruelensis. Rev. Gen. Bot.. 88, 33-42. MCIVER, S., AND SIEMICKI, R. 1977. Observations on larval Aedes aegypti CL.) as scavengers. Mosquito Neuas, 37, 519-521. NILSSON, C. 1983. Coprophagy in larval Culiseta begrothi (Diptera: Culicidae). Hydrobiologiu, 98, 267269. OVERBECK, J. 1968. Bakterien im Gewisser. Un~sclr. Wiss. Tech., 68, 587-592. RAMOSKA, W., PACEY. C.. AND WATTS. S. 1981. Tests on the pathogenicity and persistence of Bacillus rhrcringiensis var. isruelensis (Serotype H-14) and Bacillus sphaericus Neide against larvae of Culex restuans Theobald. .I. Kansas Entomol. Sot.. 54, 56-60. SCHNETTER,
W.,
ENGLER,
S.,
MORAWCSIK,
J..
AND
ALY
BECKER. N. 198 I. Wirksamkeit von Boci//rf.\ thltriw giensis var. isrne/en.sis gegen Stechmiickenlarven und Nontarget - Organismen. Mitt. disc/r. Ges. u//g. angew’. Entomol., 2, 195-202. SERVICE. M. 1983. Biological Control of Mosquitoes Has it a future’? Mo.wprito News. 43, I 13- 120. STAHLY, D. P.. DINGMAN, D. W., BULLA, L. A., AND ARONSON, A. I. 1978. Possible origin and function of the parasporal crystals in Bncillus thuringiensis. Biochem. Biophys. Res. Commun.. 84, 581-588. TYRELL,
D.,
DAVIDSON,
L.,
BLJLLA,
L.,
AND RAMOSKA,
W. 1979. Toxicity of parasporal crystals. of Buci//us thuringiensis subspecies isruelensis to mosquitoes. Appl. Environ. Microbial., 38, 656-658. VANKOVA, 1. 1982. Stabilittit und Wirksamkeit eines Prsparates von Bacillus thuringiensis var. isrnelensis gegen Stechmiickenlarven im Freiland. An;. Schiidlingskd. Pjlanz. Umweltschutz, 55, 17-19. VASQUEZ-GARCIA, M. 1983. “Investigations on the Potentiality of Resistance to Bucillrrs thtrringierrsis ser. H- 14 in Culex qrrinqrcefusciatus through Accelerated Selection pressure in the Laboratory.” Thesis. Univ. of California. Riverside.
OF INVERTEBRATE
Germination
PATHOLOGY
45, l-8 (1985)
of Bacillus thuringiensis var. israelensis Gut of Aedes Larvae (Diptera: Culicidae)’ CHRISTOPH
Spores
in the
ALY
Department of Entomology. University of Califovniu, Riverside, California, Received January 3. 1984: accepted July 30, 1984
92521
In laboratory experiments, germination and growth of Bacillus thztringiensis israelensis in the gut of Aedes uegypti and A. rexans larvae (Culicidae: Diptera) was observed. The number of spores and vegetative cells in the gut of fiving larvae and in cadavers was estimated by plating homogenized larvae on selective agar plates. The number of spores per gut increased in the first 40-140 min of exposure to a maximum, and decreased in the subsequent time, demonstrating spore germination in living larvae, moribunds, and in cadavers. Twenty-four hours after the death of the larvae, a minimal amount of spores, but an increased number of vegetative cells. was found in cadavers. In A. aegypti larvae, germination and growth of B. tlutringiensis israelensis in the larval gut was photographically documented. B 1985 Academic PESS, I~C. KEY WORDS: Bacillus thrrrinpiensis israelensis; Aedes aegypti: Aedes vexans: bacterial germination.
Bacillus thuringiensis var. israelensis was originally isolated from samples collected at a temporary mosquito breeding site in Israel (Goldberg and Margalit, 1977). Protein crystals, formed by the bacterium during sporulation, are a highly effective stomach poison for mosquito larvae (Tyrell et al., 1979; Lacey, 1984). Since toxicity is selective for Nematocera larvae (Schnetter et al., 1981), and resistance against the toxin remains low in mosquito populations (Vasquez-Garcia, 1983), B. thuringiensis israelensis is considered one of the most promising biocontrol agents in mosquito control (Anonymous, 1982). However, unlike classical biocontrol agents, which are characterized by their ability to recycle in the host and to suppress host populations over time, this pathogen is unable to establish high, lasting infections among natural mosquito populations (Ramoska et al., 1981; Vankova, 1982). Mosquito control requires frequent applications, which limits the importance of this agent (Service, 1983). In order to clarify the biological association between B. thuringiensis israe-
lensis and mosquitoes
as hosts, observations about the fate of this bacterium in the gut of Aedes larvae have been initiated and are reported here. MATERIALS
AND METHODS
Bottom soil and organic debris containing eggs of Aedes vexans were collected from natural mosquito breeding areas in the Upper Rhine Valley, Federal Republic of Germany, and were stored moist at room temperature in plastic bags. After flooding approximately 600 g of the soil and organic debris with 5 liters of tap water maintained at 30°C (Horsfall, 1956), 200-400 larvae hatched within 24 hr. The larvae were reared at ambient room temperature (20”-25°C). and were fed daily with Tabimin tish food (Tetra Co. ; composition Kardatzke and Liem, 1972). Random tests showed that more than 98% of the hatching larvae were A. vexans. A. aegypti larvae from a stock colony were reared at 22”-24°C on a yeast/dog food diet. Fourthinstar larvae were obtained S-6 days after hatching. All experiments were carried out using early 4th instars. B. thuringiensis israelensis. A freezeLarvae.
’ This work was partly supported by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 1
0022-2011185 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserves
2
CHRISTOPH
dried primary powder of a strain resistant to Rifampicin and Amphotericin B (Krieg et al., 1980) with a spore content of 4 x IO’ spores/mg was prepared. Test solutions were suspended with an ultrasonic blender (Braun Co.) and pasteurized (15 min, 7O”C), and a series of decimal dilutions was made in 0.25 mM phosphate buffer, pH 7.2. One hundred microliters of each dilution was mixed in a Petri dish with approximately 8 ml of liquid nutrient agar solution (Difco), containing 50 ppm Rifampicin and 4.0 ppm Amphotericin B. Colonies were counted after 18 hr incubation at 30°C. Using this method, the titer of a spore suspension could be estimated with an accuracy of to.3 x lox spores/ml. Cell titers were estimated with the same procedure, omitting ultrasonic suspension and pasteurization; in this case, colony counts represent the number of vegetative cells plus the number of spores of a suspension. All experiments were carried out in a continuously illuminated climate chamber. The experimental containers were covered with glass plates to prevent the disturbance of the water surface by the air current. In experiments I and 2, the larvae were kept without food 1 hr prior to the addition of the spore-toxin suspension. After the exposure to the test solution, the larvae were collected in a flame-sterilized metal-mesh basket and rinsed with tap water. In order to estimate the content of B. thuvingiensis israelensis spores and cells in the gut, the larvae were transferred into glass vials containing I ml ice-cold phosphate buffer, pH 7.2, per larva. In experiments 1 and 2, the larvae were homogenized with an ultrasonic blender. In experiment 3, the larvae were pulled to pieces with flame-sterilized forceps, and were homogenized by shaking the vial on a Vortex mixer (1 min, maximum speed). After taking an aliquot for estimating the cell titer in experiment 3, the suspensions were pasteurized, diluted, and plated as described above. Surface contamination of larvae. The number of spores adhering to the larval cu-
ALY
ticula after exposure to spore-toxin suspensions was estimated in the following experiment: groups of 10 larvae each were exposed to 100 ml ice-cold water containing lo4 spores/ml. At this temperature, larvae have no feeding activity. After 0.5, 1. 3, 6, and 24 hr, three groups of larvae were collected and washed as described above; each group was transferred to 1 ml ice-cold buffer. After vigorous shaking on a Vortex mixer, the number of spores present in the suspension was estimated as described. Experiment 1. To estimate the number of spores in the gut as related to time of exposure and water temperature, two l-liter glass containers were filled with distilled water and 100 A. vexans larvae each. One container was incubated at 19.5”C, the other at 27°C. After an acclimatization period of I hr, a spore-toxin suspension was added to final concentrations of 3 x IO3 spores/ml. After 20, 40, 60, 80, 100, 140 min, and after 3, 5, and 15 hr of exposure, the feeding activity of the larvae was checked by estimating the number of larvae still moving their labral brushes: 10 larvae were collected randomly to estimate the number of spores in the gut as described. The experiment was repeated with a sporetoxin concentration of lo4 spores/ml. Experiment 2. A series of beakers was filled with 100 ml water and 30 A. aegypti larvae each. By adding a spore-toxin suspension, a concentration of 1.9 X lo4 spores/ml was adjusted in all beakers. After 0.5. 1,2,4, 6,8, 12, 17, and 24 hr, mortality was recorded, and the larvae out of two beakers were collected to estimate the number of spores within the larvae. With exposure times of 0.5, 1, 2, and 24 hr, the experiment was carried out three times (six replicates). Experiment 3. To estimate the number of spores in the gut as related to the presence of food, 20 200-ml polystyrene beakers were each filled with distilled water and 20 A. vexans larvae. In 10 beakers approximately 20 mg wheat flour per beaker was added as a suspension. In all beakers, a
GERMINATION
OF
B. thuringiensis
spore-toxin concentration of IO4 spores/ml was adjusted; larvae were added after a lhr starvation period in clear water. After 20, 40, 60, 80, 100, 315, and 440 min and after 24 hr the larvae from one pair of beakers were collected. The larvae fed with wheat flour were placed individually on microscope slides; the amount of wheat flour in the gut, visible through the transparent cuticle, was examined under a dissecting microscope (method described by Dadd, 1968); each group of larvae was transferred to 1 ml buffer and homogenized. The titer of the homogenates was estimated as described. Optical observation of spore germination in gut. To suppress the growth of other bacteria in larvae killed by B. thuringiensis israelensis, sterile solutions of 50 ppm Rifampicin and 4.0 ppm Amphotericin B were used exclusively. To decontaminate the gut content, A. aegypti larvae were exposed for 1 hr to yeast cell suspensions in the antibiotic solution. Larvae were washed and transferred to suspensions of 50 mg B. thuringiensis israelensis primary powder1100 ml solution. The high concentration of primary powder was used to obtain a synchronous death of the larvae after ingestion of large and easy to observe amounts of spores. After 30 min of exposure, larvae were again washed, and their gut content was checked under the dissecting microscope as described. Control larvae were exposed to yeast cell suspensions and killed by decapitation. Larvae were transferred to aereated particle-free antibiotic solutions; 0, 6, 12, and 24 hr after death of the larvae, three larvae killed by B. thuringiensis israelensis toxin and three control larvae were collected and dissected; the gut content was checked under a phase-contrast microscope. RESULTS
Optical observation. After 30 min of exposure to 50 mg primary powder in 100 ml water, larvae were found hanging motionless at the water surface. Examination of
ismeiensis
IN Aedes
3
the gut content under the dissecting microscope showed that the larvae had nearly completely filled their gut with primary powder (4.6 & 1 gut segments filled out of a total gut volume of 6 segments). Under the phase-contrast microscope, dark-contrasted vegetative cells were already present in the gut besides bright-contrasted, ungerminated spores (Fig. 1A). Six, twelve, and twenty-four hr after the death of the larvae, masses of vegetative B. thuringiensis israelensis cells were observed in the esophagus (Fig. 1B) as well as in the gut (Fig. 1D). No bacterial growth was observed in control larvae up to 24 hr after decapitation (Fig IC). Surface contamination. After exposure to ice-cold spore suspensions, between 112 and 222 (mean = 179, standard deviation = 29) spores were found to adhere to the cuticula of one larva. This amount represents the number of spores not removed by the washing procedure, but resuspended by shaking larvae in buffer solution. No correlation with length of exposure time was found. Since this experimental situation was not identical with the situation in experiments 1-3, e.g., no cleansing activity by the larvae, data of experiments were not corrected with respect to the surface contamination data found here. Experiment 1. The feeding activity in the presence of B. thuringiensis israelensis primary powder suspensions was checked every 20 min in the first 5 hr of exposure. A concentration of 3 x lo3 spores/ml did not cause feeding to stop within this observation time. However, after 15 hr of exposure, all larvae had stopped feeding and the mortality rate totaled 58% (19.5’C) and 67% (27°C). A level of 1 x lo4 spores/ml caused a stoppage in feeding in the majority of the larvae after 160-180 min of exposure. The mortality rate totaled 15% (19.5C) and 27% (27°C) after 5 hr, and reached 100% at both temperatures after 15 hr of exposure. The spore content of the guts reached a maximum, in both spore concentrations and at both temperatures, 80- 100 min after
4
CHRISTOPH
ALY
FIG. 1. Bacillus tlzuringiensis israrlensis spores and vegetative cells in the gut of Ardes rwgypri larvae. Larvae were kept in solutions of antibiotics, and were exposed to 50 mg primary powder of an antibiotic-resistant 5. thwingiensis israelrnsis strain per 100 ml water. 100% mortality resulted after 30 min of treatment. Bars represent 10 urn. (A). After 30 min of exposure, vegetative cells (darkcontrasted) among ungerminated spores (bright-contrasted) can be observed in the gut. (B). Vegetative cells in the esophagus,. 12 hr after death of larva. CC). Gut of control larva, killed by decapitation and kept in antibiotic solution for 24 hr. Only yeast cells and no bacteria are present within the peritrophic membrane. (D). Gut of treated larva containing masses of vegetative 5. tlturingiensis israelensis ceils within the peritrophic membrane 24 hr after death of the larva.
GERMINATION
OF B. thrrringiensis
israelensis
IN Aedes
t IminI
t (min)
FIG. 2. Average spore content of 10 Aedes vexans fourth instars exposed to 3 x IO3 spores/ml (A) or IO4 spores/ml (O), at 19.5” (A) or 27°C (B). Five hours after treatment, larvae exposed to the lower spore concentration were still feeding. Spore content was estimated by plating homogenized larvae on selective agar plates.
the start of the experiment (Fig. 2). No difference between the numbers of spores present in the gut at two temperatures was observed when the same spore concentrations were offered. Larvae treated with 3 x lo3 (1 x 104) spores/ml showed a maximal spore content of 2.1-2.4 x lo3 (2.S 2.6 x 103) spores/larva after 80-100 min; the spore content declined to 1 .O- 1.2 x lo3 (2.0 x 103) spores/larva after 5 hr, and to 1.1-1.5 X 102(1.9 x lo*) spores/larvaafter 15 hr of exposure. In the surviving larvae treated with 3 x IO3 spores/ml for 15 hr, 7.0 X 102- 1.0 X lo3 spores/larva were found. Experiment 2. After 30 and 60 min of exposure, 1.3-3.1 and 3.6-7.3 x 10) spores per larva were recorded (Fig. 3). No change in behavior or feeding activity could be observed. Two hours after treatment, almost no feeding activity was observed; some larvae showed misorientated and ineffective swimming movements, but all larvae were still alive. Titers of 3.6-9.0 x lo3 spores/larva were estimated. After 4 hr, mortality totaled 95 ? 5%, and the number of spores per larva had dropped to 2.2-3.2 x 102. Up to 24 hr after treatment, similar numbers of spores were found in the cadavers.
Experiment 3. In this experiment not only the spore content but also the number of vegetative B. thuringiensis israelensis cells in the larvae up to 24 hr after treatment was estimated; additionally, feeding rates were recorded by measuring the amount of wheat flour ingested by the
IO4
0 0 III 12
I 4
I 6 Hrs.
I 8
I IO after
I 12
I 17
I 24
treatment
FIG. 3. Average spore content of 30 Aedes aegypti fourth instars exposed to 1.9 x lo4 spores/ml (T = 24-26”(I). Four hours after treatment, 95% mortality was observed. Living larvae were removed. Bars indicate standard deviation of six replicates.
6
CHRISTOPH
larvae. After 20, 40, or 60 min of exposure, larvae had 2.8 (standard deviation = 0.6), 5.1 (SD = 0.9), or 5.6 (SD = 0.7) gut segments filled with wheat, respectively. After 40 min of exposure, 6 out of 20 larvae had guts completely filled with wheat flour ( = 6 gut segments wheat filled). After 80 min, all larvae showed completely wheat-filled guts. In the absensce of food, the maximal spore content of I. 1 X lo4 spores/larva was observed 140 min after the start of the experiment (Fig. 4). In the presence of wheat flour, the maximum was observed 40 min after the start with 5.8 x IO3 spores/gut. Twenty-four hours after the start, 100% mortality was observed in both containers; 8.3-8.8 x lo? spores were counted within the larvae. However, 3.7-4.2 x lo4 vegetative cells were found in the cadavers (Table 1). This indicates not only spore germination, but also growth of vegetative cells in the dead host. DISCUSSION
Spores of B. thcwingiensis israelensis, ingested by Aedes larvae, germinate in the gut. After the death of the larvae, vegetative cells multiply within the peritrophic membrane. Approximately 1 day after
3
I 12
6
Time
(h)
FIG. 4. Average spore content of 20 Aedes t~exans fourth instars exposed to 10“ spores/ml, either with (0) or without (0) wheat flour (T = 22°C). Twenty-four hours after treatment, 8.3 x lo* (0) and 8.8 x lo* (0) spores/larva were estimated.
ALY
death, lysis of the larval tissues leads to mixing of gut contents with fluids in the hemocoel, resulting in the presence and growth of B. thuringiensis israelerzsis cells in all parts of the body. Time of spore germinution. Results indicate that germination of ingested spores occurs in living larvae, in moribunds, and in cadavers. In experiment 1, no stoppage in feeding was observed up to 5 hr after treatment with 3 x IO3 spores/ml. Nevertheless, the number of spores present in the gut declined after 80- 100 min of exposure. Defecation of spores is unlikely. since in A. vexans larvae the contents of the gut are not mixed by peristalsis (Aly, 1983). but are only pressed caudad and defecated when more food is ingested. Since the dry weight of the contents of one gut ranges between 100 and 200 pg, and only 0.75 kg (3 x lo4 spores/ml) and 2.25 )-Lg(lo4 spores/ml) primary powder/larva was offered in experiment I, replacement of the gut content, followed by defecation of the first ingested spores, is only possible if larvae feed continuously on their fecal pellets (Nilsson, 1983). In this case, the spore content of the gut should increase and remain on a constant level. Declining numbers of spores can only be explained with spore germination in the living larva. Decline in the number of spores in the gut also continued after stoppage in feeding and death, demonstrating a continuation of spore germination in moribunds and cadavers. Filter activity of larvae. The average filtration rate of A. vexans fourth-instar larvae is 3.3 t 1.1 ml hr-t larva-‘, when larvae are kept under the conditions of experiments 1 and 3 without food (T = 22°C 1-hr prestarvation; Aly, 1983). With a population density of 1 larva/l0 ml water, filtering during 160 min or more (exp. l), the number of spores suspended in the water would have declined during the exposure time due to filtration and ingestion. In these time frames, there was also a decline in the number of spores in the gut. This was due to both the decreasing number of spores
GERMINATION
Bacillus
thuringiensis
Time of exposure (hr) 1 2
5 24
OF B. fhuringiensis
isruelensis
TABLE 1 TOTAL CELLS AND SPORESIN THE GUT OF Aedes vexans
israelensis
Total cells/gut Without food
x x x x
3.7 x 104
103 lo3 103 lo4
LARVAE~
Spores/gut
With foodb 7.8 4.2 4.5 4.2
7
IN Aedes
With foodb 4.9 2.8 7.6 8.3
x x x x
Without food
103 103 lo? 10’
x x 6.1 x 8.8 x 6.6 8.9
103 lo3 lo3 lo2
n Twenty 4th-instar larvae in 200 ml of a suspension with lo4 spores/ml. b Twenty milligrams wheat flour/200 ml water.
suspended in the water and available for ingestion and to the continuing germination of the already ingested spores. Competition with other bacteria. Without antibiotics, the comparatively large cells of B. thuringiensis israelensis were few among enormous masses of other bacteria, also developing in the cadaver. Plating homogenized larvae without pasteurization on nonselective nutrient broth agar plates revealed up to lo7 bacteria per A. aegypti cadaver 1 day after death. Although unselective plating does not give a precise estimate of bacterial populations (Overbeck, 1968), the cadaver of a mosquito larva killed by B. thuringiensis israelensis toxin cannot be considered to be a relatively exclusive growth medium for this bacterium (Stahly et al., 1978). Nevertheless, B. thuringiensis israelensis was found to be able to multiply in Aedes larvae in competition with other bacteria. Sporulation and crystal production in the host. In the present paper, only germination and vegetative growth in the host have been described. From other experiments, resporulation and crystal production also seems possible: Larget (1981) and Ignoffo et al. (1981) observed a higher toxicity of B. thuringiensis israelensis suspensions when previously killed larvae had not been removed from the experimental containers. In A. aegypti (McIver and Siemicki, 1977) and A. vexans (Aly, unpubl.), larvae have been observed feeding on larval cadavers; this behavior may act as an important link
in the epizootiology of B. thuringiensis israelensis in natural mosquito populations. ACKNOWLEDGMENTS The author acknowledges the support of the present study by W. Schnetter, the technical assistance of B. Zimmermann, and the critical review of the manuscript of E. W. Davidson and C. Lucarotti.
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GOLDBERG, L.. AND MARGALIT, J. 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergeniii. Uranotaenia Culex uni~itattas, Aedes aegypti Mosquito News, 37, 355-358.
anguiculata,
and Calex pipiens.
HORSFALL, W. R. 1956. Eggs of floodwater mosquitoes III. (Diptera: Culicidae): Conditioning and hatching of Aedes vexans. Ann. Entomol. Sot. Amer..
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IGNOFFO,C. M., GARCIA, C.. KROHA, M. J., FUKUDA, T.. AND COUCH, T. L. 1981. Laboratory tests to evaluate the potential efficacy of Bacillus thuringiensis var. israelensis for use against mosquitoes. Mosquito
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41, 85-93.
KARDATZKE, J.. AND LIEM, K. 1972. Growth of Aedes stimulans and Aedes vexans (Diptera: Culicidae) in saline solutions. Ann. Entornol. Sot. Amer. 65, 1425-1426. KRIEG. A., ENGLER, S., AND RIEGER, M. 1980. Produktion von Prgparaten auf der Basis von Bacillas tharingiensis var. israelensis mit UV inaktivierten Sporen zur biologischen Bekgmpfung von Stech-
CHRISTOPH
8
miickenlarven. Anz. Schiidlingskd. Pflun;. Umweltschutz, 53, 129-133. LACEY, L. 1984. Bacillus fhwingiensis serotype H-14 for the microbial control of mosquitoes. &r/l. Amer. Mosq. Contr. Assoc., in press. LARGET. I. 1981. &de de la rCmanence de Bucillus thuringiensis var. isruelensis. Rev. Gen. Bot.. 88, 33-42. MCIVER, S., AND SIEMICKI, R. 1977. Observations on larval Aedes aegypti CL.) as scavengers. Mosquito Neuas, 37, 519-521. NILSSON, C. 1983. Coprophagy in larval Culiseta begrothi (Diptera: Culicidae). Hydrobiologiu, 98, 267269. OVERBECK, J. 1968. Bakterien im Gewisser. Un~sclr. Wiss. Tech., 68, 587-592. RAMOSKA, W., PACEY. C.. AND WATTS. S. 1981. Tests on the pathogenicity and persistence of Bacillus rhrcringiensis var. isruelensis (Serotype H-14) and Bacillus sphaericus Neide against larvae of Culex restuans Theobald. .I. Kansas Entomol. Sot.. 54, 56-60. SCHNETTER,
W.,
ENGLER,
S.,
MORAWCSIK,
J..
AND
ALY
BECKER. N. 198 I. Wirksamkeit von Boci//rf.\ thltriw giensis var. isrne/en.sis gegen Stechmiickenlarven und Nontarget - Organismen. Mitt. disc/r. Ges. u//g. angew’. Entomol., 2, 195-202. SERVICE. M. 1983. Biological Control of Mosquitoes Has it a future’? Mo.wprito News. 43, I 13- 120. STAHLY, D. P.. DINGMAN, D. W., BULLA, L. A., AND ARONSON, A. I. 1978. Possible origin and function of the parasporal crystals in Bncillus thuringiensis. Biochem. Biophys. Res. Commun.. 84, 581-588. TYRELL,
D.,
DAVIDSON,
L.,
BLJLLA,
L.,
AND RAMOSKA,
W. 1979. Toxicity of parasporal crystals. of Buci//us thuringiensis subspecies isruelensis to mosquitoes. Appl. Environ. Microbial., 38, 656-658. VANKOVA, 1. 1982. Stabilittit und Wirksamkeit eines Prsparates von Bacillus thuringiensis var. isrnelensis gegen Stechmiickenlarven im Freiland. An;. Schiidlingskd. Pjlanz. Umweltschutz, 55, 17-19. VASQUEZ-GARCIA, M. 1983. “Investigations on the Potentiality of Resistance to Bucillrrs thtrringierrsis ser. H- 14 in Culex qrrinqrcefusciatus through Accelerated Selection pressure in the Laboratory.” Thesis. Univ. of California. Riverside.